1. Field of the Invention
This invention relates generally to the field of compound identification and, more particularly, to a novel system having the capability to rapidly, automatically, and specifically identify and quantitate any one of a set of pre-selected compounds from unknown and impure samples. The present invention is described with respect to an embodiment incorporating a mass spectrometer.
2. Prior Art
The use of mass spectrometry for the indentification of compounds and determination of their molecular structure is well known in the art. In a mass spectrometer, a sample gas is partially ionized by electron impact or other means in an ion source. For each compound in the sample, a set of fragment ions are typically formed, each one having a particular mass to charge ratio, m/e, where m is the mass of the ion in atomic mass units and e is the charge of the ion determined by the number of electrons removed therefrom by the ionization. The mass to charge ratio, m/e, is usually referred to as "mass".
The ions are separated by electric, magnetic or combined fields (in a mass analyzer) into different species according to their respective masses. In the usual arrangement of the mass analyzer, ions of one mass at a time are transmitted to a suitable detector, typically an electron multiplier, for measurement and/or recording. Usually, the mass analyzer controls are manipulated so that the m/e values are repeatedly and continuously swept over a selected mass range. A plot or tabulation of ion current or intensity vs m/e is referred to as a mass spectrum and is the basic data output from a mass spectrometer. It should be understood that references to mass peaks made herein may apply to the amplitude of said ion intensity, the integral of ion intensity with respect to m/e, or any other quantitative measure of the presence of ions. If the mass separative power or resolution of the mass analyzer is such that integral values of m/e can be separated but fractional values cannot, the technique is referred to as low resolution mass spectrometry.
One prior art example of mass analyzer control manipulation is disclosed by Krutz et al. (U.S. Pat. No. 3,642,420). Krutz et al. teaches the use of a computer to control the power supplies of a plurality of mass spectrometers. The Computer is programmed to intermittently vary the voltage of a mass spectrometer from one general area to another where known mass peaks corresponding to elements of interest are located. Once the computer has manipulated the power supply to the correct general area, it then increases and decreases the voltage within this area by very small increments so that a peak may be accurately defined. In this manner the mass peaks of the unknown compound may be located so that some independent means of identification may be used to determine the components of the unknown compound.
A second prior art example of mass analyzer control manipulation is disclosed by Laukien (U.S. Pat. No. 3,639,738). Laukien teaches the utilization of a computer to control the step-by-step scanning of a spectrum. In one embodiment spectral ranges of particular interest may be preselected so that such ranges may be repeatedly scanned. The existence or absence of a spectral line within the preselected ranges can be established through a form of statistical correlation. In a second embodiment, ranges of interest are determined by the signal amplitude present within the spectrum. Thus, the invention may be configured to repeatedly scan ranges wherein a signal exceeds a given threshold value.
Interpretation of the mass spectrum has two related but somewhat different objectives. Identification is the process of determining which (if any) compounds in a predetermined list or library are present in the sample, by means of a comparision of the sample spectra with previously recorded spectra of known pure compounds. In structure determination, part or all of the molecular structure of an unknown compound are deduced from the mass spectrum. This invention relates to the identification function of mass spectrometry (and the quantitation of the compounds identified).
The mass spectrum analysis for identification purposes may be performed manually or with the assistance of electronic analysis means. For a manual analysis, a skilled mass spectrometrist is normally required to study the data for features which suggest the possible identity of the compound sampled. Tables, computations, and application of rules of the formation of mass spectra are typically used. The final identification is usually made by a comparison of the selected sample spectrum with a published or measured spectrum for the compound identified.
Mass spectrum identification by electronic analysis means is typically accomplished by encoding or contracting the mass spectrum according to any one of a number of rules. Examples of some of the rules used include: (i) selection of n most intense peaks; (ii) selection of one or two of the most intense peaks in each mass range of 14 amu; and (iii) binary encoding (indicating presence or absence only) of all peaks. The encoded spectrum is then compared with each of a number of similarly-encoded library spectra. Based on some criterion of similarity, the compound whose library spectrum most closely resembles that of the sample is identified as the compound which was sampled. Often more than one possible identification is provided, with the final identification being left to the operator based on comparisons of complete or encoded spectra.
An example of mass spectrum identification by electronic analysis is disclosed by Hargens, et al. (U.S. Pat. No. 3,027,086). Hargens et al. takes information from a direct reading spectrograph and produces a teletype record of the percent concentration of the component elements of the sample. Essentially, the Hargens et al. invention accepts signals from the spectrograph, which indicate the spectral line intensities, computes the percent concentration of the constituent elements, and then prints a record of the computation. Of course, of particular importance to such analysis is the separate and independent provision of electrical signals from the direct reading spectrograph. That is, in the Hargens et al. utilization of a spectrograph, the spectrograph provides signals which indicate the spectral line intensity ratio of a particular alloying element to a reference element. Then, the Hargens et al. device computes the percent concentration of the elements and provides a printed record of the product of the computation.
A second prior art example of electronic analysis of waveforms is disclosed by Watkins et al. (U.S. Pat. No. 3,614,408). Used in conjunction with chromatographs, the Watkins et al. device integrates the incoming signal between selected component peaks and valleys to provide a measure of particular band components. Thus, essential to the operation of this device is a "valley" sensor which indicates the absence of spectral peaks.
Use of mass spectrometry for identification presently requires purification of the sample by physical or chemical means. One or more abundant ions from an impurity could conceivably misdirect the identification of the compound sought. The purification may be accomplished before introduction of the smaple into the apparatus, or a separative device may be attached to the mass spectrometer. External means of sample purification include extraction (using control of pH and suitable solvents to preferentially dissolve the compound(s) of interest), distillation, recrystallization and thin layer chromatography. The separative device most commonly used with a mass spectrometer is a directly interfacing gas chromatograph (GC), providing the well known gas chromatograph/mass spectrometer (GC/MS) instrument. Recently, a liquid chromatograph has been used in conjunction with a mass spectrometer for separation of the sample.
In a gas chromatograph, a sample comprised of one or more compounds is injected by a syringe or valve into a heated chamber (the flash evaporator or injector) or directly into a chromatographic column. The sample is vaporized (if it is not already a gas) and transported through the column by a suitable inert carrier gas. The column is a glass or metal tube, usually packed with a powdered support material. The tube or the support material therein is coated with an organic liquid, called the stationary phase or liquid phase. The liquid phase has the property of absorbing and desorbing each of the constituent compounds in the sample at different rates, thereby causing a different rate of slowing of each compound as it passes through the column. As a result, the different constituents comprising the sample pass through the column at different rates and emerge therefrom at different times. Under fixed operating conditions (column type and temperature, flow rate, etc.) each compound has a characteristic, reproducible retention time, or delay from injection to elution at the column outlet. In this manner, a mixture is separated into its constituent compounds. Each compound then flows into the mass spectrometer for identification.
An interfacing device or separator is usually required between the outlet of the gas chromatograph and the inlet to the mass spectrometer, because the pressure at the column outlet is typically one atmosphere, while the mass spectrometer must operate in a vacuum of the order of 10.sup.-.sup.8 atmospheres. The separator transmits a reasonable fraction of the compounds of interest while excluding most of the carrier gas. Usually, a suitable non-selective detector is used to indicate GC peaks, that is, the elution of each of the constituent compounds. At least one mass spectrum is taken as each GC peak is detected.
The principal disadvantages of the instruments known in the prior art are as follows:
a. Where the mass spectrometer is not equipped with a separative device such as a gas or liquid chromatograph, lengthy, complex and tedious sample purification methods must be employed. PA1 b. Where a separative device is used, the device itself imposes additional limitations. For example, only those constituent compounds of a sample which can be successfully separated by the separative device can be analyzed. Secondly, the operation of the separative device typically requires at least several minutes; thereby, it introduces a time delay. Moreover, the separative device increases the complexity of the instrumentation and the skill required to operate it. PA1 c. Manual interpretation of mass spectra requires a great deal of time and skill. PA1 d. Computing means for aiding the interpretation of mass spectrum used in the prior art, suffer from one or more of the following disadvantages: (i) a significant amount of operator intervention is necessary and, therefore, the interpretations are not fully automatic. In addition, a relatively high degree of operator skill is required. (ii) The computing means are typically used to make comparisons between a measured spectrum and library spectra, or characteristic peaks thereof. Such comparisons are made on the basis of an assumption that the spectrum measured is that of a pure sample. Thus, in order for the computing means to be effective, it requires purification of the sample (typically by means of a gas chromatograph). (iii) In making comparisons, the quantitative criteria of similarity typically applied by systems of the prior art do not reflect directly the probability that the identification is correct. (iv) The complexity of the comparison method requires the use of a high capability computer and/or temporary storage so that analysis can be completed after the data mseasurement is completed. Thus, the analysis cannot ordinarily be done in "real time", that is, as the measurement is being made. (v) Prior art computing means for identification of a compound from the mass spectrum typically fail to make use of some of the information available therein, including the absence or weakness of characteristic peaks and the differing significance of peaks as a function of their mass and intensity. (vi) The relatively long time required for the comparison process usually precludes an analysis of the plurality of spectra derived from mass measurement of the gas chromatograph effluent.
The present invention overcomes substantially all of the above-described limitations and shortcomings of the prior art instruments and methods. This invention enables the identification of any one of a number of a preselected "target" compounds in unknown mixtures of compounds with little or no sample purification. Thus, for one thing, it enables the elimination of a gas chromatograph or, at the least, a substantial reduction in its complexity. As a result, this invention provides a system which is less complex and less expensive than the corresponding systems of the prior art. In addition to the elimination or simplification of the gas chromatograph, the present invention further reduces the cost and complexity of compound identification by eliminating bulk data storage means. This is the result of its incorporation of means capable of real time analysis.
The present invention also enables greater specificity in the identification of compounds than that possible using the systems and methods of the prior art. This is due to the novel analysis means incorporated into the invention, means which (i) apply probabilistic techniques to the comparisons made; (ii) carry out exhaustive analysis of each spectrum measured; (iii) make use of negative information, e.g., the absence or weakness of characteristic peaks; and (iv) make use of calibration data "learned" from the invention itself. In addition, the analysis means of this invention can provide a confidence index, consistent for all target compounds, which quantitatively indicates the probability that the identification is correct.
The present invention enables automatic identification of compounds. This has the advantage of reducing to a minimum the skill and attention required of the operator. In addition, the invention enables rapid operation. For example, in applications where direct mass analysis gives satisfactory results, i.e., where chromatographic separation is not necessary, analysis of a sample can require as little as one second and is typically completed in 30 seconds, including data printout. Moreover, a greater variety of samples can be analyzed in situations where the chromatograph is eliminated than has heretofore been possible. Even in situations where some chromatographic separation is necessary, less analysis time is required than in the prior art because the degree of separation necessary to achieve satisfactory results is substantially reduced by the invention.
One further advantage of the present invention lies in its making possible the quantitation of identified compounds even when the target compound mass spectrum is largely obscured by other compounds in the mixture.
While some instruments disclosed by the prior art overcome some of the disadvantages described above, there has heretofore been no system which combines in one structure all of the features and advantages found in the present invention.